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Lime witches’-broom (WBDL) is a devastating disease of Mexican lime trees in southern Iran.‘CandidatusPhytoplasma aurantifolia’ is associated with the disease. Understanding the pattern of colonization and spread of ‘Ca. P. aurantifolia’ within lime trees is critical for its accurate detection during the latent period of the disease. In the present study, the distribution pattern of the WBDL phytoplasma in graft inoculated lime seedlings was investigated under greenhouse conditions and its seasonal variation of concentration in symptomless lime trees was evaluated in an infected orchard using qPCR assay. Repeated sampling from various tissues of graft-inoculated lime seedlings was carried out at various times post-inoculation. In the field, leaf samples were taken from symptomless trees in an infected orchard in March, August and December. Our results showed that after entrance into the lime seedlings, ‘Ca. P. aurantifolia’ moved downward into the roots as well as upward into the apical leaves. The concentration of phytoplasma in apical leaves showed an initial high followed by a decrease before establishment of infection and subsequent symptom appearance. The spread of the phytoplasma from root to the aerial parts was rather slow. Severe witches’ broom symptoms appeared on the grafted branches 10 months after grafting. At this time, the highest phytoplasma DNA concentration was found in descending order in stems and leaves of witches’ brooms, crown, various parts of the root, and stem bark and apical leaves of the symptomless branches. Results of the field experiment revealed that concentration of ‘Ca. P. aurantifolia’ cells in infected trees was highest in August compared to that in December and March. Our result would be useful for rapid detection of WBDL phytoplasma during the latent period of the disease.